Summary
Square Bin
Cell Bin
Image

Spatial Gene Expression Distribution

Spatial gene expression distribution plot shows MID count at each spot (bin200)

Tissue Plot with Spot (bin200) Colored by MID Count

1,299,371,915

Total Reads

28,830

Mean MID per Bin200

5,005

Mean Gene per Bin200

36,271,914

Unique Reads

Key Metrices

Total Reads

Total number of sequenced reads

Valid CID Reads

Number of reads with CIDs matching the mask file and with MIDs passing QC

Invalid CID Reads

Number of reads with CIDs that cannot be matched with the mask file

Discarded MID Reads

Number of reads with MID that have been discarded since MID sequence quality does not satisfy with further analysis

Clean Reads

Number of Valid CID Reads that have passed QC

Non-Relevant Short Reads

Number of non-relevant short reads

Uniquely Mapped Reads

Number of reads that mapped uniquely to the reference genome

Multi-Mapped Reads

Number of reads that mapped more than one time on the genome

Unmapped Reads

Number of reads that cannot be mapped to the reference genome

Annotated Reads

Number of reads that are aligned to transcripts of at least one gene

Unannotated Reads

Number of reads that cannot be aligned to transcript of one gene

Total Reads

100%

1,299,371,915

Valid CID Reads

77.1%

1,002,113,195

Clean Reads

85.1%

852,423,959

Uniquely Mapped Reads

65.7%

560,226,716

Annotated Reads

74.8%

419,110,974

Unique Reads

8.7%

36,271,914

Sequencing Saturation

91.3%

382,839,060

Unannotated Reads

25.2%

141,115,742

Multi-Mapped Reads

24.8%

211,588,387

Unmapped Reads

9.5%

80,608,856

Non-Relevant Short Reads

14.9%

149,689,236

Invalid CID Reads

20.9%

271,798,958

Discarded MID Reads

2.0%

25,459,762

Information

Species

mouse

Tissue

tongue

Reference

mouse

FASTQ

V350066444_L04_read_1,V350066578_L02_read_1,V350066578_L03_read_1,V350066578_L04_read_1

Sequencing

Total Reads

Total number of sequenced reads

Total Q30

Ratio of bases with quality value exceeded 30 in the sequenced reads

CID Q30

Ratio of CID (Coordinate ID) bases with quality value exceeded 30

MID Q30

Ratio of MID (Molecule ID) bases with quality value exceeded 30

Total Reads

1,299,371,915

Total Q30

82.55%

CID Q30

88.33%

MID Q30

85.65%

Annotation

Exonic

Number of reads that mapped uniquely to an exonic region and on the same strand of the genome

Intronic

Number of reads that mapped uniquely to an intronic region and on the same strand of the genome

Intergenic

Number of reads that mapped uniquely to an intergenic region and on the same strand of the genome

Transcriptome

Number of reads that mapped to a unique gene in the transcriptome. These reads are considered for MID counting. (Transcriptome = Exonic + Intronic)

Antisense

Number of reads mapped to the transcriptome but on the opposite strand of their annotated gene

Exonic

360,653,186

Intronic

58,457,788

Intergenic

141,115,742

Transcriptome

419,110,974

Antisense

122,900,040

RNA Mapping

Clean Reads

Number of Valid CID Reads that have passed QC

Uniquely Mapped Reads

Number of reads that mapped uniquely to the reference genome

Multi-Mapped Reads

Number of reads that mapped more than one time on the genome

Unmapped Reads

Number of reads that cannot be mapped to the reference genome

Clean Reads

852,423,959

Uniquely Mapped Reads

560,226,716

Multi-Mapped Reads

211,588,387

Unmapped Reads

80,608,856

Tissue

DNB Under Tissue

Number of DNBs under tissue coverage region

mRNA-Captured DNBs Under Tissue

Number of DNBs under tissue that have captured mRNA

Genes Under Tissue

Number of detected gene under tissue coverage

Number of MID Under Tissue Coverage

Number of MID under tissue coverage

Fraction MID in Spots Under Tissue

Fraction of MID under tissue over total unique reads (MID Under Tissue / Unique Reads)

Reads Under Tissue

Number of reads with position prior to filtration under tissue coverage

Fraction Reads in Spots Under Tissue

Fraction of mapped reads under tissue over total mapped reads (Mapped Reads in Spots Under Tissue / (Valid CID Reads + Discarded MID Reads))

DNB Under Tissue

26,483,222

mRNA-Captured DNBs Under Tissue

9,481,859

Genes Under Tissue

22,848

Number of MID Under Tissue Coverage

21,190,353

Fraction MID in Spots Under Tissue

58.42%

Reads Under Tissue

512,269,047

Fraction Reads in Spots Under Tissue

49.85%

Sequencing Saturation

(left) Plot of the sequencing saturation as a function of sequencing depth(bin200)

(middle) Plot of the median number of genes detected per bin as a function of sequencing depth(bin200)

(right) Plot of the unique reads detected per bin as a function of sequencing depth(bin200)

Tissue Square Bin Statistics

Bin Size

The size of Bin which is the unit of aggregated DNBs in a squared region (i.e. Bin 50 = 50 * 50 DNBs)

Mean Reads (per bin)

Mean number of sequenced reads divided by the number of bins under tissue coverage

Median Reads (per bin)

Median number of sequenced reads divided by the number of bins under tissue coverage (pick the middle value after sorting)

Mean Gene Type (per bin)

Mean number of unique gene types divided by the number of bins under tissue coverage

Median Gene Type (per bin)

Median number of unique gene types divided by the number of bins under tissue coverage

Mean MID (per bin)

Mean number of MIDs divided by the number of bins under tissue coverage

Median MID (per bin)

Median number of MIDs divided by the number of bins under tissue coverage

Bin Size

Mean Reads
(per bin)

Median Reads
(per bin)

Mean Gene Type
(per bin)

Median Gene Type
(per bin)

Mean MID
(per bin)

Median MID
(per bin)

7,660

7,041

168

169

316

312

47,126

46,658

773

798

1,949

1,995

100

183,608

190,867

2,171

2,308

7,595

7,973

150

403,044

429,347

3,660

3,971

16,672

17,848

200

696,964

760,490

5,005

5,541

28,830

31,675

Bin 20

(left) Scatter plot of MID count and gene number in each bin

(right) Violin plots show the distribution of deduplicated MID count and gene number in each bin

(bottom) Univariate distribution of MID count, gene numbers, and DNB numbers with rug along the x axis

Bin 50

(left) Scatter plot of MID count and gene number in each bin

(right) Violin plots show the distribution of deduplicated MID count and gene number in each bin

(bottom) Univariate distribution of MID count, gene numbers, and DNB numbers with rug along the x axis

Bin 100

(left) Scatter plot of MID count and gene number in each bin

(right) Violin plots show the distribution of deduplicated MID count and gene number in each bin

(bottom) Univariate distribution of MID count, gene numbers, and DNB numbers with rug along the x axis

Bin 150

(left) Scatter plot of MID count and gene number in each bin

(right) Violin plots show the distribution of deduplicated MID count and gene number in each bin

(bottom) Univariate distribution of MID count, gene numbers, and DNB numbers with rug along the x axis

Bin 200

(left) Scatter plot of MID count and gene number in each bin

(right) Violin plots show the distribution of deduplicated MID count and gene number in each bin

(bottom) Univariate distribution of MID count, gene numbers, and DNB numbers with rug along the x axis

Clustering

(left) Clustering spots (bin200) under tissue covered with the Leiden algorithm

(right) UMAP projections of spots (bin200) colored by automated clustering. Same color is assigned to spots that are within shorter distance and with similar gene expression profile

Tissue Plot with Spots (bin200)

UMAP Projection of Spots (bin200)

Cell Bin Statistics

Cell Count

Number of cells

Mean/Median Cell Area

Mean/Median cell area (pixel)

Mean/Median DNB Count

Mean/Median number of DNBs that have captured-mRNAs per cell

Mean/Median Gene Type

Mean/Median genes per cell

Mean/Median MID

Mean/Median MID count per cell

Cell Count

24,410

Mean Cell Area

240

Median Cell Area

215

Mean DNB Count

161

Median DNB Count

137

Mean Gene Type

119

Median Gene Type

105

Mean MID

224

Median MID

187

Cell Bin

(left) Scatter plot of MID count and gene number in each bin

(right) Violin plots show the distribution of deduplicated MID count and gene number in each bin

(bottom) Univariate distribution of MID count, gene numbers, DNB numbers, and cell area (pixel) with rug along the x axis

Clustering

(left) Clustering spots (cell bin) under tissue covered region with the Leiden algorithm

(right) UMAP projections of spots (cell bin) colored by automated clustering. Spots in the short distance are assigned with the same color have similar gene expression profile.

Tissue Plot with Spots (cell bin)

UMAP Projection of Spots (cellbin)

Image Information

Manufacture

motic

Model

MoticamProS5 Lite M

Scan Time

2022-1-20 15:1:34

Overlap

0.1

Exposure Time (ms)

11.795

Scan Rows

Scan Columns

FOV Height

2,035

FOV Width

2,436

Stain Type

ssDNA

Stitched Image

Image Name

SS200000154TR_F5_20221124_180305_1.1.3

Stitching

Template Source Row No.

The row number of the template FOV used for predicting the entire template

Template Source Column No.

The column number of the template FOV used for predicting the entire template

Global Height

Height of the stitched image

Global Width

Width of the stitched image

Template Source Row No.

Template Source Column No.

Global Height

18,523

Global Width

17,787

ImageQC version

QC Pass

Whether the image(s) passed imageQC quality check

Track Line Score

Reference score for track line detection

Clarity Score

Reference score for image clarity

Good FOV Count

Number of FOVs that have at least one track dot detected

Total FOV Count

Total number of FOVs

Stitching Score

Reference score for stitching

Tissue Segmentation Score

Reference score for tissue segmentation

Registration Score

Reference score for aligning image with gene expression matrix

ImageQC Version

1.1.3

QC Pass

Pass

Track Line Score

Clarity Score

Good FOV Count

Total FOV Count

Stitching Score

Tissue Segmentation Score

100

Registration Score

Registration

ScaleX

The lateral scaling between image and template

ScaleY

The longitudinal scaling between image and template

Rotation

The rotation angle of the image relative to the template

Flip

Whether the image is flipped horizontally

Image X Offset

Offset between image and matrix in x direction

Image Y Offset

Offset between image and matrix in y direction

Counter Clockwise Rotation

Counter clockwise rotation angle

Manual ScaleX

The lateral scaling based on image center (manual-registration)

Manual ScaleY

The longitudinal scaling based on image center (manual-registration)

Manual Rotation

The rotation angle based on image center (manual-registration)

Matrix X Start

Gene expression matrix offset in x direction by DNB numbers

Matrix Y Start

Gene expression matrix offset in y direction by DNB numbers

Matrix Height

Gene expression matrix height

Matrix Width

Gene expression matrix width

ScaleX

1.003

ScaleY

1.003

Rotation

0.858

Flip

True

Image X Offset

6,077

Image Y Offset

1,488

Manual ScaleX

Manual ScaleY

Manual Rotation

Counter Clockwise Rotation

Matrix X Offset

2.00

Matrix Y Offset

7.00

Matrix Height

26,458

Matrix Width

26,452

Horizontal Image Stitching Deviation Heatmap

Image stitching deviation in the horizontal axis, a lighter color indicates better stitching (the heatmap and frame of axes have been adjusted with the registration parameters)

Vertical Image Stitching Deviation Heatmap

Image stitching deviation in the vertical axis, a lighter color indicates better stitching (the heatmap and frame of axes have been adjusted with the registration parameters)